Overexpression of PpSnRK1α in Tomato Promotes Fruit Ripening by Enhancing RIPENING INHIBITOR Regulation Pathway

Abstract

As a conserved kinase complex, sucrose non-fermenting-1-related protein kinase 1 (SnRK1) is a major regulator of plant growth and development. In our previous study, overexpression of MhSnRK1 in tomato (Solanum lycopersicum L.) modified fruit maturation: the transgenic fruit ripened earlier than the wild type (WT). However, the mechanism by which fruit maturation is regulated by SnRK1 is not clear; therefore, the test materials used were the transgenic tomato lines (OE-1, OE-3, and OE-4) overexpressing the coding gene of peach [Prunus persica (L.) Batsch] SNF1-related kinase α subunit (PpSnRK1α). The activity of SnRK1 kinase in transgenic tomato lines OE-1, OE-3, and OE-4 was higher than that in the WT at different periods of fruit development; in the pink coloring period the SnRK1 kinase activity increased the most, with 23.5, 28.8, and 21.4% increases, respectively. The content of starch and soluble sugars in red ripe transgenic fruit significantly increased, while the soluble protein and titratable acid content decreased significantly. We also found that the tomatoes overexpressing PpSnRK1α matured approximately 10 days earlier than the WT. Moreover, the yeast-two-hybrid assay showed that PpSnRK1α interacted with the MADS-box transcription factor (TF) SIRIN, which acts as an essential regulator of tomato fruit ripening. The BiFC technology further validated the location of the PpSnRK1α interaction sites within the nucleus. The quantitative real-time PCR analysis showed that RIN expression was up-regulated by PpSnRK1α overexpression; the expression of RIN-targeted TF genes NOR and FUL1 increased during different stages of fruit development. The expression of key genes, ACS2, ACS4, and E8, in ethylene synthesis also changed accordingly, and the ethylene emitted by the red ripe fruit increased by 36.1–43.9% compared with the WT. These results suggest that PpSnRK1α interacts with SIRIN, increasing the expression of RIN, thereby regulating the expression of downstream ripening-related genes, finally promoting fruit ripening.

Genome-Wide Characterization of Snf1-Related Protein Kinases (SnRKs) and Expression Analysis of SnRK1.1 in Strawberry

“…For instance, a tomato overexpressing PpSnRK1α caused higher sugar content and matured approximately days earlier than the WT. It was demonstrated that PpSnRK1α interacted with the MADS-box transcription factor SIRIN, increasing the expression of RIN, regulating the expression of downstream ripening-related genes and promoting the fruit ripening [37]. Here, the high expression level of FvSnRK1.1 during fruit development suggested that FvSnRK1.1 was probably involved in strawberry fruit maturation, but the detailed mechanism requires in-depth study.…”

“…In addition, FvSnRK1.1 transcript accumulated in tissue-specific manner. It has been documented that overexpression of SnRK1 could increase the fruit starch and soluble sugar and promote fruit ripening [35][36][37]. For instance, a tomato overexpressing PpSnRK1α caused higher sugar content and matured approximately days earlier than the WT.…”

Sugar Signaling During Fruit Ripening

“…Increased carbon metabolism in the leaves could explain the higher available carbon in the fruits stored as starch and soluble sugars ( Wang et al., 2012 ). A second study overexpressing Prunus persica SNF1-related kinase α subunit ( PpSnRK1α ) in Sy12f tomato allowed to go deeper in understanding the molecular mechanism by which SnRK1 regulates fruit ripening ( Yu et al., 2018 ). By yeast-two-hybrid experiment and BiFC assay, positive interaction was detected between PpSnRK1α and RIN.…”

“…By yeast-two-hybrid experiment and BiFC assay, positive interaction was detected between PpSnRK1α and RIN. Furthermore, in PpSnRK1α overexpressing fruits, RIN expression, together with the levels of other TFs directly controlled by RIN, were enhanced, suggesting a regulation by SnRK1 both at the post- and transcriptional level ( Yu et al., 2018 ).…”

Peach PpSnRK1 Participates in Sucrose-Mediated Root Growth Through Auxin Signaling

“…Approximately 1.0 g of fresh roots was weighed and minced with a high-throughput tissue grinder. Subsequently, 1 ml of pre-cooled extraction buffer was added, and the extraction was performed as described previously (Yu et al, 2018). The AMARA polypeptide served as the substrate (Zhang et al, 2009;Debast et al, 2011), and SnRK1 activity was measured with the Universal Kinase Activity Kit (R&D Systems, Minneapolis, MN, United States).…”